<Originals>Biochemical studies on the corneal epithelial cell-derived stimulator of collagen gel contraction by keratocytes

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概要

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• Background. In corneal wound healing, contraction of the stromal scar tissue changes the shape of the cornea and corneal refraction. In this study, to understand the mechanism of corneal wound contraction, the effect of corneal epithelial cells on collagen gel contraction by keratocytes was investigated. And a contraction-stimulating factor derived from corneal epithelial cells was purified and identified. Methods. The cultured medium of rabbit corneal epithelial cells was collected and used as an epithelial cell-conditioned-medium (ECCM). Subcultured rabbit keratocytes were embedded in a collagen gel and collagen gel contraction by them was investigated. The effect of ECCM on collagen gel contraction by keratocytes was also investigated. The contraction-stimulating factor in ECCM was purified through acetone precipitation, affinity chromatography (heparin), gel filtration and reverse-phase chromatography. The amino acids sequence of a contraction-stimulating factor was analyzed. Results. The diameter of the collagen gel decreased in proportion to the number of keratocytes. Collagen gel contraction by keratocytes was enhanced by the addition of ECCM in a dose-dependent manner. The amino acids sequence of the contraction-stimulating factor was 90% homologous to 32-KDa glycoprotein : SPARC (secreted protein which is acidic and rich in cystein). Immunoblotting analysis confirmed that SPARC was contained in ECCM. Collagen gel contraction by keratocytes was enhanced by the addition of purified SPARC in a dose-dependent manner. Conclusions. These findings demonstrated that keratocytes contracted the collagen gel and corneal epithelial cells secreted a factor which stimulated collagen gel contraction by keratocytes. It was also suggested that the contraction-stimulating factor derived from corneal epithelial cells might be SPARC.

• 近畿大学の論文

著者

• Hibino T

  Department Of Ophthalmology Kinki University School Of Medicine

• Hibino Tsuyoshi

  Department Of Cardiology Gifu Prefectural Tajimi Hospital

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